In recent years, the measurement of biologically active components in a living body, such as insulin, growth hormones, prolactin, gastrin, adrenocorticotrophic hormones, thyroid hormones, angiotensin, and the like, has been recognized as having important significance in the diagnosis, therapy, and prevention of human diseases or in various functional tests. As a result, such measurements have recently come to be carried out very frequently.
However, as all these biological components are present in very small quantities, they cannot be measured by a conventional chemical method and are measured by an immunochemical method, such as by a radioimmunoassay (hereinafter referred to as RIA) which enables extraordinarily highly sensitive measurement.
However, the RIA method is unsuitable for measuring components of the human body as a matter of daily routine in general medical institutions, since it requires special instruments utilizing isotopes and the operation thereof is complicated and inconvenient.
Heretofore, a well known simple method for measuring biological components of the human body has comprised sensitizing a carrier composed of blood cells, polystyrene latex, kaolinite, bentonite, active charcoal, crystalline cholesterin, and the like with an antigen or antibody, and then reacting the carrier with the antibody or antigen present in the sample and causing an immunochemical agglutination reaction or an agglutination inhibition reaction. Particularly, a method using blood cells as the carrier is much preferred because of its high sensitivity.
The blood cell used as a carrier for the abovementioned immunochemical measurement is usually prepared from blood cells obtained from various animals without any chemical treatment or blood cells first treated with an appropriate fixing agent such as formaldehyde, pyruvinaldehyde, hydrogen peroxide, or the like, and then treated with a binding agent such as tannic acid, bisdiazobenzidine, 1,3-difluoro-4,6-dinitrobenzene or the like.
The animals from which the blood cell is obtained are generally mammals, e.g. cattle, horses, sheep, rabbits, humans, etc. or birds, e.g. chicken, pigeons, turkeys, etc., though other animals including reptiles such as crocodiles and snakes and amphibians such as frogs, etc., and marine animals such as dolphins may also be used.
Although the blood cell obtained from these animals can be used in its untreated state, in most cases, a fixed blood cell, treated with the above mentioned fixing agent is used because an untreated blood cell has poor strength in its natural form tending to hemolyze during mechanical or chemical treatments.
The term "blood cells" in this specification means the material component in the blood consisting mainly of red blood cells which includes untreated blood cells without any treatment and the said fixed blood cells. The quantity of blood cells is represented by the apparent volume of blood cells obtained by the centrifugation of blood.
These blood cells have to be treated with the binding agent as mentioned above before they are used as a carrier. It is hard to bind antigens or antibodies to blood cells which are not treated with the binding agent and even if they are bound, no uniform product can be obtained, resulting in failure to obtain a carrier having a stable sensitivity. On the other hand, a blood cell treated with a binding agent exhibits improved bonding power on the surface of the blood cell enhancing the binding to the antigen or antibody, so that a carrier with a better immunochemical agglutination reaction or agglutination inhibition reaction is obtained. For instance, when tannic acid is used as a binding agent according to the conventional method, a suspension of blood cells in a buffer solution (blood cell concentration 2 to 8%) is mixed with an equal volume of a solution of binding agent in the said buffer solution and both components are allowed to react at 37.degree. to 56.degree. C for 30 to 60 minutes. The said solution of tannic acid is usually a concentration of 1/20,000 to 1/40,000, the representation 1/20,000 of said tannic acid solution indicates that the 20,000 ml solution was prepared by adding 1 g of the solute to the solvent. Other cases follow suit.
As above, if blood cells are treated with tannic acid, about 0.3 to 2.5 mg of tannic acid is bound to 1 ml of blood cells. The blood cells have a sensitivity of about 100 ng/ml in the measurement of human serum albumin (hereinafter referred to as HSA) with the blood cells as a carrier. This value of sensitivity is comparatively high as compared with that with other types of carriers, such as the fine particles of polystyrene latex, other than the blood cells. Nevertheless, the value is no more than 1/10 to 1/10,000 as compared with the sensitivity of the RIA method.
Accordingly, in immunochemical agglutination reactions or agglutination inhibition reactions, it is impossible to obtain a sensitivity comparable to that of a RIA method by using a conventional carrier. In other words, the measurement of such high sensitivity required a novel carrier.
As a highly sensitive and highly specific carrier used for an immunochemical measurement the carrier should have the following properties: when the carrier is sensitized with an antigen or antibody corresponding to the object substance of the measurement, any slight quantity of antigen or antibody present in a living body as biological components can readily bring about an immunochemical reaction with the antibody or antigen with which the carrier was sensitized and, based on this reaction, the sensitized carrier undergoes a complete agglutination reaction; however, in the event that the immunochemical reaction does not take place, no spontaneous agglutination occurs.